As a consequence of the antiparallel arrangement of the two strands of the double helix and the 5'-3' polarity of DNA polymerases, DNA replication is intrinsically asymmetric. Only one parental strand at the replication fork can be continuously replicated: the other - the lagging strand - is discontinuously synthesized via the iterative generation, processing and ligation of Okazaki fragments to generate an intact daughter strand. Furthermore, the genome is not a homogeneous substrate. Significant variation exists in protein binding, the potential to form hard-to-replicate secondary structures, and the presence of unrepaired damage to the DNA template. Despite this heterogeneity, both leading- and lagging-strand synthesis must proceed efficiently and with extremely high fidelity throughout the entire genome to avoid genome instability, a hallmark of both the development and progression of cancer. The overall goal of this project is to determine how the replisome - the highly conserved multi-protein complex that carries out DNA replication - coordinates the activities of polymerases, helicases and nucleases to ensure faithful replication in the face of replication asymmetry, genomic heterogeneity and DNA damage. We have recently developed innovative protocols that allow the direct analysis of lagging-strand synthesis intermediates generated in vivo. We will apply and modify these techniques as part of a combined genetic, biochemical and genomic approach in which we will use the budding yeast Saccharomyces cerevisiae to: 1 - Establish the roles of conserved helicases and nucleases in the generation of an intact lagging strand. 2 - Define the mechanisms underlying polymerase dynamics during lagging-strand synthesis, and determine the phenotypic consequences of perturbing this dynamic behavior. 3 - Elucidate the role of polymerase dynamics in the bypass of damaged DNA bases. The expected overall impact of this proposal is a significant advance in our fundamental mechanistic understanding of DNA replication - a process that is both essential for life and invariably subverted in cancer. Because both the mechanism and machinery of DNA replication are highly conserved throughout eukaryotes, our findings in S. cerevisiae will be directly applicable to human health.